We propose to develop a novel dual FRET molecular beacons technology for living cell detection and analysis of cancer. Molecular beacons are dual-labeled oligonucleotide probes with a stem-loop hairpin structure. Hybridization of molecular beacons with target mRNAs corresponding to cancer markers results in fluorescence of the cell. Thus, cancer cells (bright) can be distinguished from normal cells (dark). However, the conventional design of molecular beacons may induce a significant amount of false positives in cancer cell detection due to probe degradation by nucleases and non-specific interactions. To overcome this difficulty, we have developed the dual FRET molecular beacons approach in which a pair of molecular beacons with respectively donor and acceptor fluorophores hybridizes to adjacent regions of the same target mRNA and results in a FRET signal upon proper excitation, which is readily differentiable from non-FRET false-positive signals due to probe degradation and non-specific probe opening. In our Phase I STTR studies, we have demonstrated that, using dual FRET molecular beacons in living cell mRNA detection, false positive signals can be significantly reduced. We have also developed new molecular beacon delivery methods with high efficiency and fast kinetics for live-cell studies, and examined the sensitivity and specificity of detecting Kras and survivin mRNAs in living cells. In Phase II STTR studies, we will demonstrate the quantitative capability of molecular beacons in detecting and analyzing cancer genes in living cells. We will use dual FRET molecular beacons to detect the up-regulation of specific genes and compare the mRNA levels detected using molecular beacons and RT-PCR. To further increase the detection sensitivity and specificity, we will use molecular beacons to target multiple sites on the same mRNA molecule and target multiple tumor markers in the same pancreatic cancer cells. We will demonstrate the capability of molecular beacons to detect mutant mRNAs in fixed or live cells and the sensitivity of detecting a small number of cancer cells in a sample. The goals are to develop the dual FRET molecular beacons technology for early cancer detection and diagnosis, and to commercialize this technology for a wide range of biomedical applications including cancer analysis, drug discovery, and in vivo detection of gene expression in basic biological studies. [unreadable] [unreadable]